PyroMark Q24 Advanced Reagents

For advanced methylation and mutation quantification in long sequence runs using Pyrosequencing

Advanced technology, software, and chemistry for long sequence runs

Quantitative methylation analysis at consecutive CpG or CpN sites

Improved quantification of sequence variations at any sequence position

PyroMark Q24 Advanced Reagents feature advanced Pyrosequencing chemistry to provide even better real-time sequence-based detection and quantification than before. PyroMark Q24 Advanced Reagents are optimized for use with PyroMark Q24 Advanced Systems and are highly suited for analyzing any kind of sequence variation, particularly DNA methylation at CpG or CpN sites, complex mutations, or for de novo sequencing applications such as microbial typing. For long-read Pyrosequencing runs which may require larger volumes of nucleotides, such as de novo sequencing or methylation analyses we recommend using the PyroMark Q24 Advanced CpG Reagents.

PyroMark Q24 Advanced Reagents are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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Long de novo sequencing runs.|Analysis of 16 CpG sites in a long sequence run.|Improved methylation quantification in homopolymers.|Quantitative mutation analysis in long sequence runs.|

Read length is a critical factor for analysis of unknown sequences, often limiting the reliable analysis to 40–80 bases. The improved chemistry and algorithm of PyroMark Q24 Advanced significantly increases the possible read length and provides higher accuracy. PyroMark Q24 (upper panel) and PyroMark Q24 Advanced (lower panel) were used for de novo sequencing using the same assay. Blue bars indicate reliably detected bases; yellow indicates bases that might have been determined correctly but should be checked by the user; red indicates unreliable readings.|PyroMark Q24 Advanced increases both read length and reliability of methylation analysis at positions later in the sequence. This example demonstrates 135 nucleotide dispensations and the accurate analysis of 16 different CpG positions in a single PyroMark Q24 Advanced CpG reaction. To accurately analyze all of these sites with PyroMark Q24, one would need to run 3 separate assays.|Quantification of CpG methylation directly following a T homopolymer or between T homopolymers is especially challenging. Methylation levels are determined by the ratio of converted C nucleotides to unconverted C nucleotides following bisulfite treatment, and since the C to T conversion often leads to long T homopolymer stretches in an amplicon, this scenario occurs often. PyroMark Q24 Advanced enables accurate quantification of CpG position after or between T homopolymers.This example shows the analysis of a CpG site within a stretch of 8 T nucleotides.|Since mutations and single nucleotide polymorphisms (SNPs) are rarely in directly neighboring positions, common Pyrosequencing chemistry might require separate assays for analysis of more than one mutation site. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of additional mutations within one run. This example shows the analysis of a 10:90 mixture of wild-type and mutated EGFR. Even after 60 dispensations, the analysis is exact.|

Longer Pyrosequencing runs and improved accuracy

PyroMark Q24 Advanced features improved chemistry and instrument operation algorithms that significantly increase the assay read length and accuracy of the base calling functionality, enabling easy analysis of long de novo sequencing runs. Assay read length was previously limited by background peaks and reduced light signals in the sequencing reaction. The updated chemistry and algorithms of PyroMark Q24 Advanced reduce these background peaks, thereby increasing read length and reliability. Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single PyroMark Q24 Advanced reaction (see figure Long de novo sequencing runs).

Improved methylation analysis at any position

PyroMark Q24 Advanced enables improved methylation quantification in long sequence runs at any sequence position. Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy, methylation quantification is highly reliable throughout the entire sequencing run (see figure Analysis of 16 CpG sites in a long sequence run).

Improved sequencing accuracy in homopolymers

Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure Improved methylation quantification in homopolymers).

Analysis of mutations over long sequences

PyroMark Q24 Advanced also provides reliable quantification of multiple polymorphisms in a single assay. Since single nucleotide polymorphisms (SNPs) and other mutations are often not located close to one another, traditional Pyrosequencing chemistry usually requires separate assays for each mutation site to be analyzed. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of more than one mutation or SNP in the same run (see figure Quantitative mutation analysis in long sequence runs).

Principle

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents feature updated Pyrosequencing chemistry to enable analysis of complex mutations, methylation analysis in epigenetic research, resistance typing, and microbial identification. These reagents are optimized for use with PyroMark Q24 Advanced Systems.

Procedure

Fast and easy sample preparation

Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. Whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, or polymorphisms in forensic samples of mitochondrial DNA, the PyroMark Q24 Advanced enables powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.

Important: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents cannot be used with PyroMark Q24 Software. PyroMark Gold Q24 Reagents cannot be used with PyroMark Q24 Advanced Software. Using the incorrect reagent and software combination will result in incorrect reagent dispensations and lead to inaccurate results.